The present invention relates to a newly isolated strain of Klebsiella oxytoca No. 19-1 which is capable of producing .alpha.-cyclodextrin in high proportion, nearly close to 100 percent, from starch.
Cyclodextrins (cycloamyloses, cyclomaltaoses, Schardinger dextrins, CDs) are cyclic, nonreducing oligossccharides composed of glucose units linked by .alpha.-1,4 glycosidic bonds. They are called .alpha.-, .beta.-, or .gamma.- cyclodextrin depending on the number of glucose units: 6, 7 or 8, respectively.
Cyclodextrins have been shown to act as host molecules, having a torus in the molecule capable of forming inclusion complexes with various kinds of organic compounds. This specific ability has been widely used in the field of food, pharmaceuticals, agricultural chemistry, cosmetics, and perfumes.
Cyclodextrins are produced from starch and related compounds by cyclomaltodextrin glucanotransferase (E.C.2.4.1.19: CGTase). Some microorganisms, such as Bacillus marcerans, Bacillus megaterium, Bacillus stearothermophilus, Bacillus circulans, Bacillus spp., Klebsiella pneumoniae M5a1, Micrococcus sp., and Thermoanaerobacter sp., are also known to produce CGTase.
Although all CGTase produced by these microorganisms can convert starch to cyclodextrins, their characteristics is different from each other. Accordingly, even if a bacterium is known for a typical strain which is producing .alpha.-cyclodextrin, it does not produce .alpha.-cyclodextrin only, but produces another types of cyclodextrins at the same time. It is, therefore, still required to complicated purification process in preparation of each cyclodextrin in high degree of purity.
In the conventional process, raw starch is usually liquefied by heating in the presence or absence of starch--hydrolyzing enzyme, and CGTase is added to liquefied starch for the synthesis of cyclodextrins. Since the distribution of produced cyclodextrins can be controlled by reaction or process conditions, there have been trials to increase the yield of a special cyclodextrin. For example, in the formation of cyclodextrin from starch by CGTase, the addition of organic compounds such as n-decanol is known to lead to an increase in .alpha.-cyclodextrin yield. But in this case, the effect of n-decanol on .alpha.-cyclodextrin yield is not significant to all kinds of CGTase: the use of n-decanol seems not to be favorable when one attempts to produce .alpha.-cyclodextrin with B. macerans CGTase (Starch/Starke, 41,417-420, 1989). In addition to, it was reported that organic solvents such as toluene, ethanol, n-butanol, and propanol increased the production yield of cyclodextrins in the conventional process. However, the use of toxic solvents like toluene and decanol seems to be undesirable since cyclodextrins produced in the presence of these solvents are prohibited in food, cosmetics, and pharmaceuticals.
Since .beta.-cyclodextrin is readily separated from the reaction mixture without any treatment with organic solvents and its inclusion complexes can be easily prepared owing to its low solubility in water, .beta.-cyclodextrin among the three common types of cyclodextrins, is more widely used and developed for applications. For practical use, however, cyclodextrins should be available at a reasonable price, much effort has been focused on finding a suitable CGTase for the efficient production of .beta.-cyclodextrin through screening of microorganisms.
As a consequence, the price of .beta.-cyclodextrin dropped drastically during the last decade. But cyclodextrins can not be used interchangably because of their different property, and .alpha.- as well as .gamma.-cyclodextrin is still relatively expensive material for applications in industry, since the purification of .alpha.- and .gamma.-cyclodextrin is more complicated than that of .beta.-cyclodextrin (for example, gel filtration processes) and often accomplished by precipitation with organic solvent.
.alpha.-Cyclodextrin may be classified as dietary fiber and is effective as a calorie substitute for weight control. Therefore, its extensive application is expected in the food and pharmaceutical industries. It is desirable to develop a novel CGTase that produces .alpha.-cyclodextrin in high proportion, nearly close to 100 percent from starch, so that the price of .alpha.-cyclodextrin can be more suitable for applications by simplifying the manufacturing processes of .alpha.-cyclodextrin without an additional gel filtration process or treatment with harmful organic solvent.
Accordingly, it is an object of the present invention to provide an enzyme which converts starch to .alpha.-cyclodextrin in very high proportion, nearly close to 100 percent, rather than another types of cyclodextrins.
It is another object of the present invention to provide a microorganism which is able to produce an enzyme capable of converting starch to .alpha.-cyclodextrin in high proportion, nearly close to 100 percent, rather than another types of cyclodextrins.